H. William Detrich

A model for cleavage plane determination in early amphibian and fish embryos.

Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.

OVERVIEW OF THE ZEBRAFISH SYSTEM

Publisher Summary The consensus of the biologists in attendance at the Third Cold Spring Harbor Conference on Zebrafish Development and Genetics is that the zebrafish has now “arrived” as a viable compelling genetic system for the study of vertebrate development. Novel contributions by the zebrafish system have been made and will continue to be made at an accelerating rate. The zebrafish embryo provides numerous opportunities to examine cellular processes in early development. For example, one can culture cells from embryos in vitro for mechanistic studies of cell signaling pathways, analyze gene, and protein expression in situ , perturb development using physical and chemical treatments or by ectopic expression of dominant negative proteins, and assay lineage commitment by explant assay. The roles of cell movements and the cytoskeleton in embryonic axis formation are particularly amenable to analysis. Given that some of these processes occur prior to the activation of the zygotic genome, maternal-effect mutants may prove to be especially informative in revealing the molecular players.

Evolutionary mutant models for human disease.

Although induced mutations in traditional laboratory animals have been valuable as models for human diseases, they have some important limitations. Here, we propose a complementary approach to discover genes and mechanisms that might contribute to human disorders: the analysis of evolutionary mutant models in which adaptive phenotypes mimic maladaptive human diseases. If the type and mode of action of mutations favored by natural selection in wild populations are similar to those that contribute to human diseases, then studies in evolutionary mutant models have the potential to identify novel genetic factors and gene-by-environment interactions that affect human health and underlie human disease.

The Major Adult α-Globin Gene of Antarctic Teleosts and Its Remnants in the Hemoglobinless Icefishes CALIBRATION OF THE MUTATIONAL CLOCK FOR NUCLEAR GENES

The icefishes of the Southern Ocean (family Channichthyidae, suborder Notothenioidei) are unique among vertebrates in their inability to synthesize hemoglobin. We have shown previously (Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella, L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 1817–1821) that icefishes retain inactive genomic remnants of adult notothenioid α-globin genes but have lost the gene that encodes adult β-globin. Here we demonstrate that loss of expression of the major adult α-globin, α1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus) results from truncation of the 5′ end of the notothenioid α1-globin gene. The wild-type, functional α1-globin gene of the Antarctic yellowbelly rockcod, Notothenia coriiceps, contains three exons and two A + T-rich introns, and its expression may be controlled by two or three distinct promoters. Retained in both icefish genomes are a portion of intron 2, exon 3, and the 3′-untranslated region of the notothenioid α1-globin gene. The residual, nonfunctional α-globin gene, no longer under positive selection pressure for expression, has apparently undergone random mutational drift at an estimated rate of 0.12–0.33%/million years. We propose that abrogation of hemoglobin synthesis in icefishes most likely resulted from a single mutational event in the ancestral channichthyid that deleted the entire β-globin gene and the 5′ end of the linked α1-globin gene.

Molecular pedomorphism underlies craniofacial skeletal evolution in Antarctic notothenioid fishes

BackgroundPedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. Despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. Notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing Antarctic climate. Notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy.ResultsWe compared craniofacial skeletal development in two pelagic notothenioids, Chaenocephalus aceratus and Pleuragramma antarcticum, to that in a benthic species, Notothenia coriiceps, and two outgroups, the threespine stickleback and the zebrafish. Relative to these other species, pelagic notothenioids exhibited a delay in pharyngeal bone development, which was associated with discrete heterochronic shifts in skeletal gene expression that were consistent with persistence of the chondrogenic program and a delay in the osteogenic program during larval development. Morphological analysis also revealed a bias toward the development of anterior and ventral elements of the notothenioid pharyngeal skeleton relative to dorsal and posterior elements.ConclusionsOur data support the hypothesis that early shifts in the relative timing of craniofacial skeletal gene expression may have had a significant impact on the adaptive radiation of Antarctic notothenioids into pelagic habitats.

Binding of glycerol by microtubule protein

Abstract When microtubules are purified by polymerization and depolymerization in a buffer containing glycerol, some glycerol becomes bound to the microtubule protein and is not removable by gel filtration or by prolonged dialysis. Both 6s tubulin and larger aggregates containing tubulin and accessory proteins bind glycerol. The 6s fraction has associated with it about 5 moles of glycerol per mole of tubulin dimer; 3 moles are exchangeable upon polymerization-depolymerization and 2 moles are not. The aggregate fraction has associated with it about 22 moles of glycerol per mole of tubulin dimer; approximately 11 moles are exchangeable and 11 moles are not.

Molecular and morphological phylogenies of the Antarctic teleostean family Nototheniidae, with emphasis on the Trematominae

Four independent molecular data sets were sequenced in order to solve longstanding phylogenetic problems among Antarctic teleosts of the family Nototheniidae. The anatomical data of Balushkin (2000) were also coded into a matrix of 106 characters in order to test the parsimony of his taxonomic conclusions. Molecular results confirm Balushkin’s Pleuragrammatinae but not his Nototheniinae. Different genes used here found the “clade A” establishing the paraphyly of the Nototheniinae sensu lato; i.e. Lepidonotothen and Patagonotothen are more closely related to the Trematominae than to Notothenia. The genus Notothenia is paraphyletic and Paranotothenia should become Notothenia. Previously no molecular data set could assign a reliable position for the genus Gobionotothen. For the first time robust results are obtained for the phylogeny among the Trematominae. Trematomus scotti is the sister-group of all others, then Trematomus newnesi emerges, then Trematomus eulepidotus. Among the crown group, three clades emerge: 1: Trematomus hansoni + Trematomus bernacchii + Trematomus vicarius; 2: Trematomus pennellii + Trematomus lepidorhinus + Trematomus loennbergii; 3: Trematomus (Pagothenia) borchgrevinki + Trematomus nicolai. Pagothenia should become Trematomus to make the genus Trematomus monophyletic. The Trematomus tree found here did not match the topology obtained with Balushkin’s morphological matrix. The tree shows that the tendencies shown by some trematomines to secondarily colonize the water column are not gained through common ancestry.

The genome sequence of the Antarctic bullhead notothen reveals evolutionary adaptations to a cold environment

BackgroundAntarctic fish have adapted to the freezing waters of the Southern Ocean. Representative adaptations to this harsh environment include a constitutive heat shock response and the evolution of an antifreeze protein in the blood. Despite their adaptations to the cold, genome-wide studies have not yet been performed on these fish due to the lack of a sequenced genome. Notothenia coriiceps, the Antarctic bullhead notothen, is an endemic teleost fish with a circumpolar distribution and makes a good model to understand the genomic adaptations to constant sub-zero temperatures.ResultsWe provide the draft genome sequence and annotation for N. coriiceps. Comparative genome-wide analysis with other fish genomes shows that mitochondrial proteins and hemoglobin evolved rapidly. Transcriptome analysis of thermal stress responses find alternative response mechanisms for evolution strategies in a cold environment. Loss of the phosphorylation-dependent sumoylation motif in heat shock factor 1 suggests that the heat shock response evolved into a simple and rapid phosphorylation-independent regulatory mechanism. Rapidly evolved hemoglobin and the induction of a heat shock response in the blood may support the efficient supply of oxygen to cold-adapted mitochondria.ConclusionsOur data and analysis suggest that evolutionary strategies in efficient aerobic cellular respiration are controlled by hemoglobin and mitochondrial proteins, which may be important for the adaptation of Antarctic fish to their environment. The use of genome data from the Antarctic endemic fish provides an invaluable resource providing evidence of evolutionary adaptation and can be applied to other studies of Antarctic fish.

Do the hemoglobinless icefishes have globin genes

Abstract Among piscine taxa, the Antarctic icefishes (family Channichthyidae) prosper in the absence of erythrocytes and hemoglobin, a unique condition among adult vertebrates. The genomes of three icefish species and four red-blooded notothenioid species were probed using α- and β-globin cDNA from the red-blooded Antarctic fish Notothenia coriiceps . High-stringency hybridization signals with the α-globin probe, but none with the β-globin probe, on genomic DNAs of both hemoglobinless and red-blooded fishes suggest that icefishes retain remnants of α-globin genes in their genomes but have lost the gene that encodes for β-globin, either through deletion or through rapid mutation. Northern blot analysis of major hematopoietic and non-hematopoietic tissues shows that the α-globin-related sequences of icefishes are nonexpressed derivatives of the α-globin genes of their red-blooded relatives. Mechanisms leading to the hemoglobinless phenotype are discussed in relation with the expression of myoglobin in the Channichthyidae family.

Microtubule assembly in cold-adapted organisms: Functional properties and structural adaptations of tubulins from Antarctic fishes☆

Fishes native to the coastal waters of the Antarctic have adapted to habitat and body temperatures in the range -1.8 to +2 degrees C. Their cytoplasmic microtubules, unlike those of mammals and temperate poikilotherms, have evolved to assemble efficiently at these low temperatures. To learn about the underlying molecular adaptations, my laboratory is studying microtubule proteins [tubulin alpha beta dimers and microtubule-associated proteins (MAPs)] and tubulin genes from several Antarctic fishes, including the rockcods Notothenia coriiceps and Gobionotothen gibberifrons. We find that the assembly-enhancing adaptations of the fish microtubule proteins are intrinsic to the tubulin subunits themselves. Furthermore, microtubule formation by Antarctic fish tubulins is strongly entropy driven, due in part to an increased reliance, relative to tubulins from other species, on hydrophobic interactions. Based on analyses of tubulin polypeptides and cDNAs, we suggest that the structural adaptations of Antarctic fish tubulins most likely involve alterations in the primary sequences of tubulin isotypes. With respect to neural beta tubulins from other vertebrates, for example, the class II beta-tubulin isotype of N. coriiceps brain contains seven unique amino acid substitutions and one novel insertion in its 446-residue primary sequence. Most of these changes are located in a structural domain that forms contacts between tubulin dimers during microtubule assembly and would be expected to enhance polypeptide flexibility, thereby facilitating addition of tubulin to microtubule ends. The acidic carboxy-terminal tails of the alpha and beta tubulins, by contrast, appear not to be sites of cold adaptation of polymerization. We have also found that brain and egg tubulins from Antarctic fishes differ strikingly in their polymerization efficiencies, which demonstrates, in agreement with the multitubulin hypothesis, that tissue-specific tubulin isoforms can possess distinct functional properties. Thus, study of microtubule proteins from organisms, such as the Antarctic fishes, that have adapted to extreme thermal regimes should contribute significantly to an understanding of the quaternary interactions that control microtubule assembly in all eukaryotes.